Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Researchers at the Earlham Institute utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation.
The researchers identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. They show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation.
Breakdown of novel transcripts identified using ONT long reads and TALON custom transcriptome annotation
Cassette exons are previously unannotated positions. *CPAT assessment of CDS coding probability (CP ≥ 0.364). **Novel junctions are previously unannotated junctions identified between existing exonic parts. All novel assessments are relative to Gencode v29 human transcriptome annotation
This work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.